Avidin dimers effective in increasing the concentration of radioactive biotin in pretargeted radioimmunotherapy

ABSTRACT

Dimers of avidin and streptavidins (diavidins) are described wherein the linker is suberate, which in turn, is bound to different functional groups (—NH2 o-COOH) of avidin. As compared to avidin, the diavidins have shown the ability to increase the amount of labelled biotin on the target, when used in an in vitro pretargeting test using supported human tenascin, the biotinylated anti-tenascin monoclonal antibody (Mab-B), avidin/diavidin, and biotin- 3 H. The use of such diavidins is also described in cancer diagnosis and anticancer therapy based on the three-step pretargeted radioimmunotherapy procedure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/506,159,filed Sep. 1, 2004, now allowed; which is a U.S. national stage under 35U.S.C. 371 of Application No. PCT/IT03/00135, filed Mar. 6, 2003, whichclaims priority benefit of Italian Application No. RM2002A000128, filedMar. 8, 2002; the contents of each of which are incorporated byreference herein.

FIELD OF THE INVENTION

The invention described herein relates to derivatives of avidin whichare useful in the diagnosis and treatment of tumours, and particularlyin the so-called three-step pretargeting method.

The invention described herein relates to modified avidins which areuseful for use in human and animal diagnosis and therapy, andparticularly for the diagnosis and treatment of pathological conditionssuch as tumours.

The invention described herein relates to the technical field of thepreparation of medicaments and diagnostic means and provides compounds,methods for their preparation, methods for their use, and compositionscontaining them which are suitable for industrial application in thepharmaceutical field.

The invention described herein provides compounds, compositions andmethods which are useful in diagnostic and therapeutic medicament, asimage acquisition techniques, and treatments for pathological conditionsof organs and tissues.

In particular, but not exclusively, the present invention relates to thefield of tumour therapy by means of radiopharmaceuticals.

BACKGROUND OF THE INVENTION

Tumour therapy is mainly implemented by means of the use of substancesaimed at killing the tumour cells. This can be achieved with cytotoxicsubstances which have to enter the tumour cell in order to exert theirfull effect, or by means of treatment of the tumour cells with radiationwith sufficient energy to kill the cell. In both cases, there is theproblem of delivering the substance as selectively as possible to thetarget cell, so as to avoid possible damage to the surrounding healthycells. In the case of radiopharmaceuticals, i.e. of substances bearingradioactive portions, the problem of selectively delivering the activepart (that is to say, the radioactive portion) to the tumour target,avoiding the spread of radionuclide in the body or in the healthy cellssurrounding the tumour, is of particular concern.

One particularly effective method for tumour detection and therapy isdescribed in patent EP 0 496 074. The protocol of this patent has beenapplied to the so-called Pretargeted Antibody-Guided Radioimmunotherapy(PAGRIT) of brain tumours. In this method, avidin is injected into thehuman subject, after the biotinylated anti-tenascin monoclonal antibody(Mab-B), to remove any free Mab-B, not bound to the tumour, from thebloodstream by forming complexes with it that are effectively eliminatedby the liver (chase effect). An infusion of streptavidin is thenadministered for the purposes of obtaining better avidination of thetumour compared to that obtainable with avidin, whose permanence in theblood is too short compared to that of streptavidin.

Though the system has shown positive clinical responses (Cremonesi M.,et al., 1999; Paganelli G., et al., 1999; Paganelli G., et al., 2001),one major limiting factor consists in the strong immune response causedby streptavidin (Paganelli G., et al., 1997). For the purposes ofovercoming these two obstacles, i.e. the high degree of immunogenicityof streptavidin and the rapid clearance of avidin, avidins have beenused which are chemically modified by covalently binding polyoxyethyleneglycol (PEG) chains to avidin, with various levels of derivatisationbased on the use of straight or branched PEGs of different molecularweights. Preliminary studies have revealed that, with the increase inthe degree of functionalisation of avidin with PEG (hereinafter referredto as pegilation), there is an increase in the plasma half-life ofavidin, a reduction in immunogenicity, and an improvement in thespecific biodistribution of the substance in relation to the tumour.

Since the ability of avidin-PEG to bind to Mab-B biotin is reduced bypegilation, the result is a reduction in the potency of the derivatives(Chinol M. et al., 1998).

A solution to this problem has been proposed in patent application WO94/23759, filed in the name of Immunomedics, where avidin multipolymersare described based on the chemical derivatisation ofhigh-molecular-weight molecules, preferably greater than 5,000 Da, suchas dextrane, proteins, and polycarboxylic acids. But none of the fivemultimers effectively described in the patent has been characterised interms of its potency of action in the pretargeting procedure or in otherprocedures.

As demonstrated in the invention described herein, the general conceptof multimerisation (also including dimerisation), given in theabove-mentioned patent application WO 94/237599, fails to providecomplete and sufficient instructions for the average technician infinding a generic avidin multimer capable of fulfilling the necessaryrequirements in the application of the three-step pretargeting method.In fact, different diavidins, obtained using different bifunctionalcross-linkers, though possessing the same ability to bind free biotin,differ in their potency when assayed in vitro in three-steppretargeting, to the extent that, in certain cases, they prove to becompletely inefficacious.

This observation indicates that the multimerisation of avidin does notautomatically produce useful functional products, but that biologicalcharacterisation is necessary for the choice of a potentiated moleculesuitable for pretargeting.

SUMMARY OF THE INVENTION

It has now been found that by binding two molecules of avidin with abifunctional linker, capable of binding the amino and/or carboxy groupsof avidin, selected from disuccinimidyl suberate (dimer hereinafterreferred to as diavidin 1) and PEG diamine with molecular weight 3400(dimer hereinafter referred to as diavidin 2), two avidin dimers areobtained which fulfil the requisites for use in the tumour treatmentmethod known as PAGRIT.

Thus the objects of the invention described herein are an avidin dimerin which two molecules of avidin are bound via the —NH₂ groups by meansof a suberate and an avidin dimer in which two molecules of avidin arebound via the —COOH groups by means of polyethylene glycol with amolecular weight of 3400.

Further objects of the invention described herein are pharmaceuticaland/or diagnostic compositions containing the above-mentioned diavidins.

Other objects of the present invention are the use of diavidins asmedicaments or diagnostic agents for pathological conditions of organsand tissues, and particularly for the preparation of medicaments usefulfor the therapy or diagnosis of tumours.

These and other objects related to the present invention will beillustrated in detail here below, also by means of experimentalexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chromatographic profile of diavidin 1 separated by gelfiltration (a), diavidin 2 separated by gel filtration (b), diavidin 3separated by gel filtration (c), protein standards for calibration (d),purified diavidin 1 (e), purified diavidin 2 (f) or purified diavidin 3(g).

FIG. 2 shows the ability of avidin, diavidin 1, diavidin 2 or diavidin 3to bind biotin by the HABA (4-hydroxy-azobenzene-2′-carboxylic acid)method.

FIG. 3 illustrates schematically an in vitro pretargeting assay.

FIG. 4 shows the in vitro pretargeting assay with avidin, diavidin 1,diavidin 2 or diavidin 3 binding either to monoclonal antibody ST2146 orST2077.

FIG. 5 shows the chromatographic profile of the crosslinking mixture atthe end of the reaction (a), purified distreptavidin (b) or streptavidin(c).

FIG. 6 shows streptavidin or distreptavidin binding to biotin.

DETAILED DESCRIPTION OF THE INVENTION

As intended in the present invention, avidin means both avidin andstreptavidin, but the case in which streptavidin is used as particularembodiment of the present invention, this fact will be specified.

Diavidin 1 was prepared by reacting avidin with disuccinimidyl suberate(DSS), having N-hydroxysuccinimidyl (NHS ester) as the active ester, DSSbeing a homobifunctional cross-linker reactive in binding the —NH₂ groupof avidin.

Diavidin 2 and diavidin 3 (negative control) were generated using PEGdiamine (PEG (NH₂)₂) with a molecular weight of 3400 and polyethyleneglycol-disuccinimidylpropionic acid [PEG(SPA)₂] with a molecular weightof 3400, respectively, as homobifunctional cross-linkers.

Due to the slower elimination of streptavidin compared to avidin fromthe circulation, distreptavidin are a particular embodiment of thepresent invention. The longer half-life is crucial to achieve themaximum increase in efficiency of avidins. The protocol for streptavidincross-linking was similar to the one used for diavidin 1 production.

The pharmaceutical or diagnostic compositions according to the inventiondescribed herein contain at least one of the diavidins described here.The diavidin will be in a mixture with suitable vehicles and/orexcipients commonly used in pharmacy, such as those described inRemington's Pharmaceutical Sciences Handbook, latest edition. Thecompositions according to the present invention will contain anefficacious amount of diavidin.

Preferred examples of pharmaceutical compositions are those that permitparenteral and locoregional administration. Pharmaceutical compositionssuitable for the purpose are solutions, suspensions, or lyophilisedforms to be reconstituted at the time of use.

As regards the use of the diavidins according to the present invention,these are particularly suitable for the preparation of medicaments ordiagnostic means for the diagnosis or therapy of pathological conditionsof tissues, such as, for example, tumours, by means of the techniqueknown as pretargeting with antibodies, and for this reason are alsosuitable for in-vitro pretargeting techniques. In one realisation by wayof an example, the pretargeting technique is implemented with abiotinylated anti-tenascin antibody, preferably a monoclonal antibody.

Suitable forms for the industrial application of the present inventionare also kits for diagnosis or therapy, particularly the radiotherapy oftumours, such as, for example, is described in EP 0 496 074; in thestudy by Paganelli C., et al., 1999; U.S. Pat. No. 5,968,405; andrelated literature.

A further object of the invention described herein is a kit for tumourtherapy or diagnosis, particularly by means of radioactivity, forexample, with the pretargeting method, preferably three-step,characterised in that at least one of the components of said kitcontains a diavidin. In said kit, one preferred biotinylated antibody isan anti-tenascin antibody, and even more preferably a monoclonalantibody.

The following examples further illustrate the invention.

EXAMPLE 1 Diavidin 1

1 ml of avidin solution, 300 μM M in PBS, pH 7.4, was mixed with 25 μlof DSS (from Pierce) 25 mM in DMSO (avidin:DSS ratio 1:2). The mixturewas incubated for 2 hours at 0° C. before blocking the reaction with 50μl of Tris 1M, pH 8.0. The choice of the aforesaid reaction ratio wasbased on preliminary tests using ratios from 1:1 to 1:10. The reactionscheme is as follows:

EXAMPLE 2 Diavidin 2

1 ml of avidin, 450 μM in PBS, pH 7.4, was mixed with 120 μl of PEG(NH₂)₂ (from Shearwater Corp.), 9 mM in H₂O, and 50 μl of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-HCl (EDAC) 260 mM in DMSO(avidin:PEG ratio 1:2.5 approx.) and left to react for 2 hours atambient temperature. At the end of this period 50 μl of Tris 1 M, pH8.0, were added and the mixture was submitted to gel filtration. Theavidin:PEG ratio was investigated over a range from 1:1 to 1:10 at areaction pH from 4.0 to 8.0. The value of the PEG:avidin ratio in thepurified diavidin 2 end product was 0.9, using the method described bySims et al., 1980. In brief, diavidin 2 was diluted to 300 μM in water,250 μl of 5% BaCl₂ in HCl 1N were added to a volume of 1 ml, and then250 μl of a solution prepared by mixing 1.27 g of I₂ in 100 ml of KI 2%.

The mixture was incubated for 15 minutes and then the absorbance readingwas taken at 535 nm. The standard curve was obtained with PEG(NH₂)₂. Thereaction scheme for diavidin 2 is as follows:

EXAMPLE 3 Diavidin 3

1 ml of avidin 150 M in PBS, pH 7.4, was mixed with 20 μl of PEGdisuccinimidyl-propionate (SPA-PEG-SPA) 20 mM in H₂O (avidin:PEG ratio1:3.5) and left to react for 2 hours at 0° C. The reaction ratio wasselected on the basis of preliminary tests conducted with ratios rangingfrom 1:2 to 1:10. The value of the PEG:avidin ratio in the purifieddimer, as determined using the method developed by Sims et al., asabove, was 3:1. The reaction scheme is as follows:

The diavidin yield in the three reactions described in Examples 1, 2 and3 was approximately 20-30%. On increasing the amount of the threelinkers in the reactions, greater final amounts of avidin oligomers wereobtained (trimer, etc., not shown), with difficulties in chromatographicseparation as a result. The reaction mixtures were analysed on aSuperdex 200-10/30 gel filtration column, while the purification of theproducts was done on a Superdex 200-16/60 column. The chromatographyprofiles of the reaction mixtures for diavidin 1, 2 and 3 are shown inFIGS. 1 a, b and c, respectively. The molecular weights of a series ofstandard proteins (calibration) are indicated at the respective elutiontimes. The calibration of the column is shown in FIG. 1 d: dextrane blue(Vo), ferritin (444 KDa), aldolase (158 KDa), albumin (67 KDa), andribonuclease (14 KDa) were used.

The purified avidin dimers are presented in FIGS. 1 e, f and g. Thesamples were separated on Superdex 200-10/15 at a flow rate of 0.5ml/min. (a-d) and 1 ml/min (e-g) in PBS on the Jasco HPLC systemconnected up to a 280 nm spectrophotometer.

EXAMPLE 4 Distreptavidin

1 ml of streptavidin (300 μM in PBS, pH 7.4) was mixed with 25 μl of DSS(25 mM in DMSO) at a streptavidin:DSS ratio of 1:2 and incubated for 2hrs at 0° C. before the reaction was quenched with 50 μl 1 M TRIS, pH8.0. A total of 4 reaction conditions were tested with a ratio ofstreptavidin: DSS ranging from 1:1 to 1:10. We selected the abovedescribed ratio of 1:2. The reaction scheme is analogous to the onesreported in the previous Examples.

The chromatographic profile of the crosslinking mixture at the end ofthe reaction for distreptavidin is shown in the FIG. 5 a. The purifieddistreptavidin is shown in the FIG. 5 b and streptavidin in FIG. 5 c.The samples were analyzed on a Superdex 200-10/15 column at a flow of 1ml/min in PBS on a Jasco HPLC system connected to a spectrophotometermeasuring the absorbance at 280 nm.

Determination of the Ability of Diavidins to Bind Biotin

To compare the ability of avidin and diavidin to bind biotin the HABA(4-hydroxy-azobenzene-2′-carboxylic acid) method was used. Avidin anddiavidins were all in a concentration corresponding to 3 μM of 67 KDaavidin monomer, in 0.1 M phosphate, 0.4 mM HABA at pH 7.0. Biotindissolved in phosphate was then added to a final concentration rangingfrom 0 to 20 μM and the absorbance was measured at 500 nm.

The ability to bind biotin was assessed as the biotin concentrationnecessary to displace 50% of bound HABA.

Biotin 5 μM approx. was capable of displacing 50% of HABA both withavidin and with the three diavidins (FIG. 2), from which it can bededuced that the diavidins conserve the total number of binding sitesafter the cross-linking. The biotin-binding properties of diavidin arecomparable to those of avidin.

In Vitro Pretargeting Assays

To test the ability of diavidins to increase the amount of radiolabelledbiotin binding to tenascin via the biotinylated anti-tenascin monoclonalantibody (Mab-B), the in vitro pretargeting assay schematicallyillustrated in FIG. 3 was used.

In brief, a 96-well plate was adsorbed with 0.5 μg/well of humantenascin (Tn—C) for 16 hours at 4° C. After three washings with PBS and0.1% Tween 20, the residual adsorbent sites in the wells were blockedwith PBS, 2% BSA, 2% and 0.1% Tween 20, for 1 hour at ambienttemperature. Two biotinylated anti-tenascin monoclonal antibodies(ST2146 or ST2077) were then incubated for 2 hours in the wells, at thesaturating concentration of 10 μg/ml. After washing as above, avidin ordiavidin were incubated in duplicate in the wells at increasingconcentrations. Lastly, a saturating amount of 5 μmol of biotin-3H (1.6TBq/mmol) was incubated for 1 hour in each well. After washings, theplate reading was taken in a β-counter. As shown in FIG. 4, for the twoMAbs used, diavidin 1 and diavidin 2 produce an increase in bound biotincompared to avidin; diavidin 3 shows no increase with MAb ST2146 orshows a reduction of binding ability with MAb ST2077.

As compared to avidin, diavidin 2 at the concentration of 2.5 μg/mlshows an increase in the amount of bound biotin-3H by a factor of 2.1(mean of 3 experiments) with Mab ST2077. For diavidin 1 the increase wasby a factor of 1.6 (mean of 6 experiments), whereas for diavidin 3 thebinding ability was lower (90%) compared to the avidin monomer.

From these experiments it can be concluded that both the length of thelinker and the binding sites involved in the diavidin dimer affect theactivity of the dimer in pretargeting mediated by biotinylatedantibodies.

Distreptavidin proved to be more potent than streptavidin in vitro asshown in FIG. 6.

Microtiter 96 well plates were coated with 0.5 μg/well of human TnC for16 hrs at 4° C., washed 3 times with PBS, 0.1% Tween 20 and then blockedfor unspecific binding with PBS, 2% BSA, and 0.1% Tween 20. Thebiotinylated anti-TnC antibody ST2146 was added at a concentration of 10μg/ml for 2 hrs. The wells were washed 3 times with PBS/Tween 20 andthereafter streptavidin or distreptavidin were added at the indicatedconcentrations. Finally, 5 μmol ³H-biotin (1.6TBq/mmol) were added, thewells incubated for 2 hrs, washed and counted in a β-counter.

As shown in FIG. 6 distreptavidin mediates increased binding ofradiolabeled biotin compared to streptavidin.

REFERENCES

-   Chinol M., Casalini P., Maggiolo M., Canevari S., Omodeo E. S.,    Caliceti P., Veronese F. M., Cremonesi M., Chiolerio F., Nardone E.,    Siccardi A. G., Paganelli G. Biochemical modifications of auidin    improve pharmacokinetics and biodistribution, and reduce    immunogenicity. British Journal of Cancer 78 (2): 189-197, 1998.-   Cremonesi M., Ferrari M., Chinol M., Stabin M. G., Grana C., Prisco    G., Robertson C., Tosi G., Paganelli G. Three-step    radioimmunotherapy with yttrium-90 biotin: Dosimetry and    pharmacokinetics in cancer patients. Eur J Nucl Med 26 (2): 110-120,    1999.-   Paganelli G., Chinol M., Maggiolo M., Sidoli A., Corti A., Baroni    S., Siccardi A G. The three-step pretargeting approach reduces the    human anti-mouse antibody response in patients submitted to    radioimmunoscintigraphy and radioimmunotherapy. Eur J Nucl Med 24:    350-351, 1997.-   Paganelli G., Grana C., Chinol M., Cremonesi M., De Cicco C., De    Braud F., Robertson C., Zurrida S., Casadio C., Zoboli S.,    Siccardi A. G., Veronesi U. Antibody-guided three step therapy for    high grade glioma with yttrium-90 biotin. Eur J Nucl Med 26 (4):    348-357, 1999.-   Paganelli G., Bartolomei M., Ferrari M., Cremonesi M., Broggi G.,    Maira G., Sturiale C., Grana C., Prisco G., Gatti M., Caliceti P.,    Chinol M. Pre-targeted locoregional radioimmunotherapy with    90Y-biotin in glioma patients: Phase I study and preliminary    therapeutic results. Cancer Biother & Radiopharm 16 (3): 227-235,    2001.-   Sims G. E. S. and Snape T. J. A method for estimation of    polyethylene glycol in plasma protein fractions. Anal Biochem 107:    60-63, 1980.

1. An avidin dimer, in which two molecules of avidin are bound via the —NH₂ groups by crosslinking with disuccinimidyl suberate.
 2. An avidin dimer, in which two molecules of avidin are bound via the —COOH groups by crosslinking with polyethylene glycol diamine with a molecular weight of
 3400. 3. The avidin dimer of claim 1, wherein the avidin is streptavidin.
 4. The avidin dimer of claim 2, wherein the avidin is streptavidin.
 5. A pharmaceutical and/or diagnostic composition containing the avidin dimer of claim
 1. 6. A pharmaceutical and/or diagnostic composition containing the avidin dimer of claim
 2. 7. A method of manufacturing a pharmaceutical and/or diagnostic composition, the method comprising mixing the avidin dimer of claim 1 with a vehicle and/or excipient to prepare a pharmaceutical and/or diagnostic composition.
 8. A method of manufacturing a pharmaceutical and/or diagnostic composition, the method comprising mixing the avidin dimer of claim 2 with a vehicle and/or excipient to prepare a pharmaceutical and/or diagnostic composition.
 9. A kit for radiotherapy or diagnosis of tumours, the kit comprising a biotinylated antibody and the avidin dimer of claim
 1. 10. The kit of claim 9 further comprising a biotinylated radiopharmaceutical.
 11. The kit of claim 9, wherein the avidin is streptavidin.
 12. A kit for radiotherapy or diagnosis of tumours, the kit comprising a biotinylated antibody and the avidin dimer of claim
 2. 13. The kit of claim 12 further comprising a biotinylated radiopharmaceutical.
 14. The kit of claim 12, wherein the avidin is streptavidin. 15-16. (canceled)
 17. A method of using the avidin dimer of claim 1 for diagnosis or treatment of pathological conditions of organs or tissues in a subject, the method comprising: (a) administering a biotinylated antibody to the subject and (b) administering an efficacious amount of the avidin dimer to the subject.
 18. A method of using the avidin dimer of claim 2 for diagnosis or treatment of pathological conditions of organs or tissues in a subject, the method comprising: (a) administering a biotinylated antibody to the subject and (b) administering an efficacious amount of the avidin dimer to the subject.
 19. A method of using the avidin dimer of claim 1 for therapy or diagnosis of tumours in a subject, the method comprising: (a) administering a biotinylated antibody specific for the tumours to the subject and (b) administering an efficacious amount of the avidin dimer to the subject.
 20. A method of using the avidin dimer of claim 2 for therapy or diagnosis of tumours in a subject, the method comprising: (a) administering a biotinylated antibody specific for the tumours to the subject and (b) administering an efficacious amount of the avidin dimer to the subject.
 21. A method of using the avidin dimer of claim 1 to pretarget a biotinylated antibody, the method comprising: (a) binding the biotinylated antibody to antigen in vitro, (b) binding avidin dimers to the bound antibody in vitro, and (c) binding radioactive biotin to the avidin dimers in vitro.
 22. A method of using the avidin dimer of claim 2 to pretarget a biotinylated antibody, the method comprising: (a) binding the biotinylated antibody to antigen in vitro, (b) binding avidin dimers to the bound antibody in vitro, and (c) binding radioactive biotin to the avidin dimers in vitro.
 23. A method of using the avidin dimer of claim 1 for treatment of disease in a subject, the method comprising: (a) administering biotinylated antibody to the subject, (b) administering an efficacious amount of said avidin dimer to the subject, and (c) administering biotinylated radiopharmaceutical to the subject.
 24. The method according to claim 23, wherein said disease is a tumour.
 25. The method according to claim 23, wherein said antibody is an anti-tenascin antibody.
 26. The method according to claim 25, wherein said anti-tenascin antibody is an anti-tenascin monoclonal antibody.
 27. A method of using the avidin dimer of claim 2 for treatment of disease in a subject, the method comprising: (a) administering biotinylated antibody to the subject, (b) administering an efficacious amount of said avidin dimer to the subject, and (c) administering biotinylated radiopharmaceutical to the subject.
 28. The method according to claim 27, wherein said disease is a tumour.
 29. The method according to claim 27, wherein said antibody is an anti-tenascin antibody.
 30. The method according to claim 29, wherein said anti-tenascin antibody is an anti-tenascin monoclonal antibody.
 31. A pharmaceutical and/or diagnostic composition containing the streptavidin dimer of claim
 3. 32. A pharmaceutical and/or diagnostic composition containing the streptavidin dimer of claim
 4. 33. A method of manufacturing a pharmaceutical and/or diagnostic composition, the method comprising mixing the streptavidin dimer of claim 3 with a vehicle and/or excipient to prepare a pharmaceutical and/or diagnostic composition.
 34. A method of manufacturing a pharmaceutical and/or diagnostic composition, the method comprising mixing the streptavidin dimer of claim 4 with a vehicle and/or excipient to prepare a pharmaceutical and/or diagnostic composition.
 35. The method according to claim 23, wherein the avidin is streptavidin.
 36. The method according to claim 27, wherein the avidin is streptavidin. 